scholarly journals Profiling Deacetylase Activities in Cell Lysates with Peptide Arrays and SAMDI Mass Spectrometry

2013 ◽  
Vol 85 (22) ◽  
pp. 10635-10642 ◽  
Author(s):  
Hsin-Yu Kuo ◽  
Teresa A. DeLuca ◽  
William M. Miller ◽  
Milan Mrksich
Keyword(s):  
Mass Spectrometry ◽  
Peptide Arrays ◽  
Cell Lysates

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Abstract TMP120: Autoantigen(s) Trigger a Robust Immune Response in Cerebral Cavernous Malformations

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Dongdong Zhang ◽  
Abhinav Srinath ◽  
Andrew J Kinloch ◽  
Robert Shenkar ◽  
Le Shen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Endothelial Cells ◽  
B Cell ◽  
Brain Tissue ◽  
Glial Cells ◽  
Plasma Cells ◽  
Normal Brain ◽  
Normal Brain Tissue ◽  
Cell Lysates ◽  
Mass Spectrometry Identification

Introduction: Previous studies have reported robust inflammatory cell infiltration, selective synthesis of IgG, B-cell clonal expansion, and deposition of immune complexes and complement within Cerebral Cavernous Malformation (CCM) lesions. Furthermore,B-cell depletion has been shown to reduce the maturation of CCM in murine models. We hypothesize that specific autoantigen(s) within the lesional milieu trigger the pathogenetic immune responses in CCMs. This study aims to identify those putative autoantigen(s) using recombinant antibodies (rAbs) derived from plasma cells found in surgical human CCM lesions. Methods: CD138 + plasma cells were laser captured from fresh frozen surgically resected human CCM lesions. Clonally expanded immunoglobulin heavy- and light-chain variable region pairs were cloned into IgG expression vectors and expressed as monoclonal antibodies. Purified rAbs were assayed by immunofluorescence with CCM lesion tissue and normal brain tissue sections. rAbs assayed by immunocytochemistry with human primary cell line were used to further define the staining pattern. The cell lysates were immunoprecipitated with rAb, after protein purification by SDS-PAGE, and analyzed by Mass spectrometry. Results: In normal brain tissue, rAbs stained endothelial cells with limited staining of glial cells. In CCM lesional tissue, rAbs stained endothelial cells, glial cells as well as structures in the acellular matrix adjacent to caverns. In cultured Human Brain Microvascular Endothelial Cells (HBMECs) and Human Astrocytes (HAs), rAbs co-localized with cytoplasmic components. After HBMEC and HA cell lysates were immunoprecipitated with rAb, a Coomassie Stain detected bands of approximately 50 kDa. Conclusions: Our results suggest that autoantigen(s) in human CCM lesions are cytoplasmic components present in lesional tissue as well as in normal brain tissue. Molecular level identification of the triggering antigen is still ongoing by mass spectrometry. Identification of the autoantigen(s) in the lesional milieu might explain the propensity of lesion development from leaky endothelium in the neuroglial parenchyma. Characterization of the autoantigen triggers will open new venues for therapy or vaccine in this disease.

scholarly journals Combining SAMDI Mass Spectrometry and Peptide Arrays to Profile Phosphatase Activities

2018 ◽  
pp. 389-403 ◽  
Author(s):  
Lindsey C. Szymczak ◽  
Che-Fan Huang ◽  
Eric J. Berns ◽  
Milan Mrksich
Keyword(s):  
Mass Spectrometry ◽  
Peptide Arrays ◽  
Phosphatase Activities

scholarly journals Combining Mass Spectrometry and Peptide Arrays to Profile the Specificities of Histone Deacetylases

ChemBioChem ◽  
2009 ◽  
Vol 10 (13) ◽  
pp. 2159-2161 ◽  
Author(s):  
Zachary A. Gurard-Levin ◽  
Joohoon Kim ◽  
Milan Mrksich
Keyword(s):  
Mass Spectrometry ◽  
Histone Deacetylases ◽  
Peptide Arrays

scholarly journals Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

scholarly journals Exosomal Proteomic Signatures Correlate With Drug Resistance and Carboplatin Treatment Outcome in a Spontaneous Model of Canine Osteosarcoma

2021 ◽  
Author(s):  
Marcus A Weinman ◽  
Stephen A Ramsey ◽  
Haley J Leeper ◽  
Jacqueline V Brady ◽  
Andrew Schlueter ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Resistance ◽  
Cell Line ◽  
Poor Responders ◽  
Osteosarcoma Cell ◽  
Chemotherapy Treatment ◽  
Liquid Biopsies ◽  
Acquired Drug Resistance ◽  
Cell Lysates ◽  
Serum Exosomes

Abstract BackgroundOsteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. MethodsIn this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. ResultsBoth cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of >300 days, and dogs with <100 days DFI revealed a proteomic signature that could discriminate between the two cohorts with high accuracy. Furthermore, when the patient’s exosomes were compared to exosomes isolated from carboplatin resistant cell lines, several putative biomarkers were found to be shared. ConclusionsThe findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

scholarly journals Intracellular protein‐drug interactions probed by direct mass spectrometry of cell lysates

2021 ◽  
Author(s):  
Rivkah Rogawski ◽  
Adi Rogel ◽  
Itai Bloch ◽  
Maayan Gal ◽  
Amnon Horovitz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Interactions ◽  
Protein Drug ◽  
Intracellular Protein ◽  
Cell Lysates ◽  
Direct Mass Spectrometry
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